Defective Bcl-2 expression in memory B cells from common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF-κB) which, in turn, promote transcriptional programs required for long-term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl-2 family which transcription is promoted by NF-κB. We found reduced Bcl-2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti-apoptotic proteins in naive B cells, mimicking T cell-dependent activation in vitro with CD40L and interleukin (IL)-21. BCL-XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B-cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl-2 protein levels in memory B cells could compromise their long-term survival, and a possible less activity of NF-κB in naive B cells, may condition an inabilityto increase BCL-XL mRNA levels, thus not promoting survival in the germinal centers.

Mutations in PIK3R1 can lead to APDS2, SHORT syndrome or a combination of the two.

Mutations in PIK3R1 gene have been associated to two different conditions: a primary immunodeficiency, called APDS2, of recent description and SHORT syndrome. 47 patients with APDS2 have been reported to date, only one of them sharing both PIK3R1-related phenotypes. Here we describe two more patients affected by APDS2 and SHORT syndrome, which highlights that this association may not be so infrequent. We recommend that patients with mutations in PIK3R1 gene should be assessed by both clinical immunologists and clinical geneticists.

Research-based flow cytometry assays for pathogenic assessment in the human B-cell biology of gene variants revealed in the diagnosis of inborn errors of immunity: a Bruton's tyrosine kinase case-study.

Introduction: Inborn errors of immunity (IEI) are an expanding group of rare diseases whose field has been boosted by next-generation sequencing (NGS), revealing several new entities, accelerating routine diagnoses, expanding the number of atypical presentations and generating uncertainties regarding the pathogenic relevance of several novel variants. Methods: Research laboratories that diagnose and provide support for IEI require accurate, reproducible and sustainable phenotypic, cellular and molecular functional assays to explore the pathogenic consequences of human leukocyte gene variants and contribute to their assessment. We have implemented a set of advanced flow cytometry-based assays to better dissect human B-cell biology in a translational research laboratory. We illustrate the utility of these techniques for the in-depth characterization of a novel (c.1685G>A, p.R562Q) de novo gene variant predicted as probably pathogenic but with no previous insights into the protein and cellular effects, located in the tyrosine kinase domain of the Bruton's tyrosine kinase (BTK) gene, in an apparently healthy 14-year-old male patient referred to our clinic for an incidental finding of low immunoglobulin (Ig) M levels with no history of recurrent infections. Results and discussion: A phenotypic analysis of bone marrow (BM) revealed a slightly high percentage of pre-B-I subset in BM, with no blockage at this stage, as typically observed in classical X-linked agammaglobulinemia (XLA) patients. The phenotypic analysis in peripheral blood also revealed reduced absolute numbers of B cells, all pre-germinal center maturation stages, together with reduced but detectable numbers of different memory and plasma cell isotypes. The R562Q variant allows Btk expression and normal activation of anti-IgM-induced phosphorylation of Y551 but diminished autophosphorylation at Y223 after anti IgM and CXCL12 stimulation. Lastly, we explored the potential impact of the variant protein for downstream Btk signaling in B cells. Within the canonical nuclear factor kappa B (NF-κB) activation pathway, normal IκBα degradation occurs after CD40L stimulation in patient and control cells. In contrast, disturbed IκBα degradation and reduced calcium ion (Ca2+) influx occurs on anti-IgM stimulation in the patient's B cells, suggesting an enzymatic impairment of the mutated tyrosine kinase domain.